The regulation of development is a fundamental biological problem. Cells must integrate external and internal information with a regulatory network that controls developmental decisions, and must orchestrate the sequence and timing of developmental events to produce specialized cell types. The nitrogen-fixing filamentous cyanobacterium Anabaena sp. PCC 7120 was chosen as a simple model of development and pattern formation. Anabaena PCC 7120 reduces N2 to ammonia in a specialized terminally differentiated cells called heterocysts. A one-dimensional developmental pattern of heterocysts and vegetative cells is established to form a multicellular organism composed of two interdependent cell types. This multicellular growth pattern, the distinct phylogeny of cyanobacteria, and the suspected antiquity of heterocyst development make this an interesting model system. Additionally, three programmed site-specific DNA rearrangements that remove DNA elements from nif and hup genes occur in heterocysts. The excision of these elements is required for nitrogen fixation and uptake hydrogenase activity. The long- term goal is to understand the signaling and regulatory pathways required for microbial development. This project is focused on two of the most interesting and distinct aspects of heterocyst development: one, the coordinated regulation of the three DNA rearrangements, and two, the control of pattern formation and the initiation of development by cell-cell communication. These studies are expected to converge in the future to allow an understanding of developmental commitment and the regulatory pathway that links the initiation of development with the final differentiated state. The two major specific objectives follow. 1) The coordinated regulation of the three DNA rearrangements is hypothesized to be linked to the developmental pathway at a common point, but to then involve different specific mechanisms for each element. This will be tested by determining the regulation involved in the activation of individual rearrangements, and by identifying genes in the developmental pathway that are required for triggering all three rearrangements. 2) The 54-bp patS gene is hypothesized to encode a diffusible peptide inhibitor that regulates pattern formation. The function of patS will be studied by genetic, molecular, and biochemical approaches. The strong selection for bypass suppressors of patS overexpression will be used to identify genes involved in patS signaling. These genes will provide the basis for understanding the signaling mechanism.